PROJECT SUMMARY Bacterial pathogens are a significant cause of morbidity and mortality that are increasingly acquiring resistance to all available antimicrobials. Of particular note is the recent emergence of antibiotic resistant strains of Staphylococcus aureus as a leading cause of bacterial infection in the United States. The identification of novel targets for therapeutic intervention is critical to our ability to protect the public health from emerging infectious threats. One promising area of potential therapeutic development involves targeting bacterial access to nutrient metal. This strategy is based on the fact that all bacterial pathogens require nutrient metal in order to colonize their hosts. Despite the fact that a variety of metals are required by bacterial pathogens during growth within vertebrates, only iron has been identified as a nutrient that is actively sequestered by the host during the innate immune response to infection. In the present application, we provide the first description of an innate immune factor, known as calprotectin, which defends against bacterial infection by chelating nutrient manganese (Mn) and zinc (Zn). Calprotectin is a pro-inflammatory molecule that is produced by neutrophils and abundant at sites of inflammation. Mice genetically deficient for the production of calprotectin are more susceptible to S. aureus underscoring the contribution of calprotectin to protection against microbial infection. These findings support metal chelation as a potent host defense against microbial invaders and describe the first Mn-chelating protein identified in vertebrates. In response to calprotectin exposure, S. aureus undergoes transcriptional reprogramming highlighted by significant alterations in the expression of anti-neutrophil toxins. Based on these fundamental new discoveries, we hypothesize that calprotectin is critical to the outcome of host-pathogen interactions. To test this central hypothesis, we propose a series of experiments aimed at understanding the mechanism and pathophysiological consequence of calprotectin-mediated metal chelation and immune cell recruitment. In these studies, we will (i) identify the biochemical and structural properties of calprotectin that are critical to function, (ii) define the effect of metal binding on the pro-inflammatory properties of calprotectin, and (iii) elucidate the impact of calprotectin on S. aureus pathogenesis. These results will lay the foundation for the creation of therapeutics based on a calprotectin scaffold to address the emerging challenge of antibiotic resistant bacterial pathogens.